Frecuencias de las principales mutaciones en el gene de la metilenotetrahidrofolato reductase en individuos afectados por defectos del tubo neural

La enzima metilenotetrahidrofolato reductase (MTHFR) cataliza la conversión del 5,10-metilenotetrahidrofolato en 5-metiltetrahidrofolato, el principal folato circulante. El gene MTHFR presenta dos polimorfismos frecuentes-C677T y A1298C. La frecuencia de la mutación C677T varía entre diferentes grupos étnicos, alrededor de 1% entre descendientes de africanos y amerindios, y de 20% entre algunos europeos y sus descendientes. Estos dos polimorfismos están asociados a las enfermedades tromboembólicas y a los defectos del tubo neural (DTN). El propósito del presente trabajo es presentar datos de un estudio sobre las frecuencias de esas mutaciones entre 119 familias con por lo menos uno afectado por DTN y también en 302 controles, provenientes de la región de Campinas, São Paulo, Brasil. No se encontraron diferencias significativas para los genotipos 677TT (χ2(3)=1.59; p=0.66) y 1298CC (χ2(3)=1.20; p=0.75). Al clasificar los DTN en defectos altos (anencefalía, cefalocele y espina bífida toracolumbar) y bajos (espina bífida lumbosacra y sacra) se observó una diferencia signicativa en la frecuencia del genotipo 677TT apenas entre los defectos bajos (χ2(1)=8.05; p=0.004). Se concluye que el genotipo 677TT es frecuente en nuestra población (cerca de 10%). Sin embargo, ésta frecuencia es mayor en los afectados por DTN bajos (27%). Los resultados también sugieren que el doble homozigoto (677TT/1298CC) parece ser una combinación de genotipos desfavorable.Asociación de Antropología Biológica de la República Argentin

Frecuencias de las principales mutaciones en el gene de la metilenotetrahidrofolato reductase en individuos afectados por defectos del tubo neural

La enzima metilenotetrahidrofolato reductase (MTHFR) cataliza la conversión del 5,10-metilenotetrahidrofolato en 5-metiltetrahidrofolato, el principal folato circulante. El gene MTHFR presenta dos polimorfismos frecuentes-C677T y A1298C. La frecuencia de la mutación C677T varía entre diferentes grupos étnicos, alrededor de 1% entre descendientes de africanos y amerindios, y de 20% entre algunos europeos y sus descendientes. Estos dos polimorfismos están asociados a las enfermedades tromboembólicas y a los defectos del tubo neural (DTN). El propósito del presente trabajo es presentar datos de un estudio sobre las frecuencias de esas mutaciones entre 119 familias con por lo menos uno afectado por DTN y también en 302 controles, provenientes de la región de Campinas, São Paulo, Brasil. No se encontraron diferencias significativas para los genotipos 677TT (χ2(3)=1.59; p=0.66) y 1298CC (χ2(3)=1.20; p=0.75). Al clasificar los DTN en defectos altos (anencefalía, cefalocele y espina bífida toracolumbar) y bajos (espina bífida lumbosacra y sacra) se observó una diferencia signicativa en la frecuencia del genotipo 677TT apenas entre los defectos bajos (χ2(1)=8.05; p=0.004). Se concluye que el genotipo 677TT es frecuente en nuestra población (cerca de 10%). Sin embargo, ésta frecuencia es mayor en los afectados por DTN bajos (27%). Los resultados también sugieren que el doble homozigoto (677TT/1298CC) parece ser una combinación de genotipos desfavorable.Asociación de Antropología Biológica de la República Argentin

Translational Data from Adeno-Associated Virus-Mediated Gene Therapy of Hemophilia B in Dogs

Preclinical testing of new therapeutic strategies in relevant animal models is an essential part of drug development. The choice of animal models of disease that are used in these studies is driven by the strength of the translational data for informing about safety, efficacy, and success or failure of human clinical trials. Hemophilia B is a monogenic, X-linked, inherited bleeding disorder that results from absent or dysfunctional coagulation factor IX (FIX). Regarding preclinical studies of adeno-associated virus (AAV)-mediated gene therapy for hemophilia B, dogs with severe hemophilia B (<1% FIX) provide well-characterized phenotypes and genotypes in which a species-specific transgene can be expressed in a mixed genetic background. Correction of the hemophilic coagulopathy by sustained expression of FIX, reduction of bleeding events, and a comprehensive assessment of the humoral and cell-mediated immune responses to the expressed transgene and recombinant AAV vector are all feasible end points in these dogs. This review compares the preclinical studies of AAV vectors used to treat dogs with hemophilia B with the results obtained in subsequent human clinical trials using muscle- and liver-based approaches

B cell–activating factor modulates the factor VIII immune response in hemophilia A

Inhibitors of factor VIII (FVIII) remain the most challenging complication of FVIII protein replacement therapy in hemophilia A (HA). Understanding the mechanisms that guide FVIII-specific B cell development could help identify therapeutic targets. The B cell–activating factor (BAFF) cytokine family is a key regulator of B cell differentiation in normal homeostasis and immune disorders. Thus, we used patient samples and mouse models to investigate the potential role of BAFF in modulating FVIII inhibitors. BAFF levels were elevated in pediatric and adult HA inhibitor patients and decreased to levels similar to those of noninhibitor controls after successful immune tolerance induction (ITI). Moreover, elevations in BAFF levels were seen in patients who failed to achieve FVIII tolerance with anti-CD20 antibody–mediated B cell depletion. In naive HA mice, prophylactic anti-BAFF antibody therapy prior to FVIII immunization prevented inhibitor formation and this tolerance was maintained despite FVIII exposure after immune reconstitution. In preimmunized HA mice, combination therapy with anti-CD20 and anti-BAFF antibodies dramatically reduced FVIII inhibitors via inhibition of FVIII-specific plasma cells. Our data suggest that BAFF may regulate the generation and maintenance of FVIII inhibitors and/or anti-FVIII B cells. Finally, anti-CD20/anti-BAFF combination therapy may be clinically useful for ITI

Safety and efficacy of factor IX gene transfer to skeletal muscle in murine and canine hemophilia B models by adeno-associated viral vector serotype 1

Adeno-associated viral (AAV) vectors (serotype 2) efficiently transduce skeletal muscle, and have been used as gene delivery vehicles for hemophilia B and for muscular dystrophies in experimental animals and humans. Recent reports suggest that AAV vectors based on serotypes 1, 5, and 7 transduce murine skeletal muscle much more efficiently than AAV-2, with reported increases in expression ranging from 2-fold to 1000-fold. We sought to determine whether this increased efficacy could be observed in species other than mice. In immunodeficient mice we saw 10- to 20-fold higher levels of human factor IX (hF.IX) expression at a range of doses, and in hemophilic dogs we observed approximately 50-fold higher levels of expression. The increase in transgene expression was due partly to higher gene copy number and a larger number of cells transduced at each injection site. In all immunocompetent animals injected withAAV-1, inhibitory antibodies to F.IX developed, but in immunocompetent mice treated with high doses of vector, inhibitory antibodies eventually disappeared. These studies emphasize that the increased efficacy of AAV-1 vectors carries a risk of inhibitor formation, and that further studies will be required to define doses and treatment regimens that result in tolerance rather than immunity to F.IX

Safety of AAV Factor IX Peripheral Transvenular Gene Delivery to Muscle in Hemophilia B Dogs

Muscle represents an attractive target tissue for adeno-associated virus (AAV) vector–mediated gene transfer for hemophilia B (HB). Experience with direct intramuscular (i.m.) administration of AAV vectors in humans showed that the approach is safe but fails to achieve therapeutic efficacy. Here, we present a careful evaluation of the safety profile (vector, transgene, and administration procedure) of peripheral transvenular administration of AAV-canine factor IX (cFIX) vectors to the muscle of HB dogs. Vector administration resulted in sustained therapeutic levels of cFIX expression. Although all animals developed a robust antibody response to the AAV capsid, no T-cell responses to the capsid antigen were detected by interferon (IFN)-γ enzyme-linked immunosorbent spot (ELISpot). Interleukin (IL)-10 ELISpot screening of lymphocytes showed reactivity to cFIX-derived peptides, and restimulation of T cells in vitro in the presence of the identified cFIX epitopes resulted in the expansion of CD4+FoxP3+IL-10+ T-cells. Vector administration was not associated with systemic inflammation, and vector spread to nontarget tissues was minimal. At the local level, limited levels of cell infiltrates were detected when the vector was administered intravascularly. In summary, this study in a large animal model of HB demonstrates that therapeutic levels of gene transfer can be safely achieved using a novel route of intravascular gene transfer to muscle

The Enhancing Effects of the Light Chain on Heavy Chain Secretion in Split Delivery of Factor VIII Gene

Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10–100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery